Conclusion

CHAPTER 6: Effect of Hot Water and Molybdenum Dips on Endogenous Polyamines and 70 kDa

5. Conclusion

The susceptibility of lemon fruit to chilling is influenced by fruit source and growing conditions. Putrescine is the dominant PA and a precursor for synthesis of spm and spd, which are higher valency PAs than put. Seemingly, the HW 53°C treatment enhances protein synthesis, including HSPs. Therefore, the more soluble-conjugated PAs can be conjugated to proteins (including 70 kDa proteins as estimated of HSP70), enzymes, bioactive compounds with antioxidant properties, to improved protection against oxidative stress. In addition, Mo treatments increased XDH activity and ROS production to levels below critical damaging threshold, which possibly signal cellular aclimation through synthesis of high affinity PAs (spermine and spermidine) via putrescine as precursor. Therefore, the combination of HW and Mo led to reduced chilling injury through enhanced protein synthesis (specifically 70 kDa protein as an estimator of HSP70)), increased ROS production and soluble-conjugate PAs to increase oxidative stress acclimation.

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List of Tables

Table 1: Effect of hot water and molybdenum postharvest dips on chilling symptoms of lemon fruit cold stored (-0.5°C) for up to 28 days

Table 2: Effect of hot water and molybdenum postharvest dips on lemon flavedo free, soluble- conjugated and membrane-conjugated polyamines over 28 days cold storage (-0.5°C) during 2007 harvest season

Table 3: Effect of hot water and molybdenum postharvest dips on lemon flavedo free, soluble- conjugated and membrane-conjugated polyamines over 28 days cold storage (-0.5°C) during 2008 harvest season

List of Figures

Figure 1: Confirmation of polyamines in lemon flavedo by chromatography. A. Polyamines authentic standards B. Flavedo free soluble polyamines C. Soluble-conjugated flavedo polyamines and D. Membrane-conjugated flavedo polyamines

Figure 2: Effect of fruit source (A-C) on free, soluble-conjugated and membrane-conjugated polyamines

Figure 3: Effect of cold storage time (A-C) on free-soluble, soluble-conjugated and membrane- conjugated PAs

Figure 4: SDS-PAGE of lemon flavedo from different growing condition over different harvest season pre-treated with hot water (47 or 53°C) and molybdenum.

Figure 5: Lemon flavedo total protein concentration over different fruit source and harvest season

Figure 5: Relationship between total soluble-conjugated polyamines and estimated HSP70 for lemon flavedo pre-treated with hot water and molybdenum before 28 days cold storage

149

Table 1

Treatments^a Ukulinga 2007^b Sun Valley Estates^c 2007

Ukulinga 2008^b Eston Estates 2008^d

Water dip (25^oC) No symptoms 0.13a No symptoms Below detection

HW 47^oC No symptoms 0.18a No symptoms Below detection

HW 53^oC No symptoms 0.09a No symptoms Below detection

1 µM Mo No symptoms 0.04ab No symptoms Below detection

1 µM Mo + HW 53^oC No symptoms 0.02ab No symptoms Below detection 10 µM Mo + HW 53^oC No symptoms 0.00b No symptoms Below detection

aTreatment effect after 28 days cold storage plus 5 days shelf-life

bUkulinga lemon fruit showed no chilling symptoms during 2007 and 2008 harvest seasonS

cSun Valley lemon fruit showed chilling symptom expressible in chilling index ,and means followed by the same letter were not significantly different at LSD(0.05) = 0.096

dEston Estates lemon did show chilling symptoms but below detection and chilling index calculation

150

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0

-10.0 20.0 40.0 60.0

80.0POLYAMINE MATHABA SPD 1.9 #1 Polyamine Mathaba UV_VIS_2 mAU

min 1

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67

8

9 WVL:254 nm

Putrecsine Spermine

Spermidine

1,6 hexanediamine

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 25.0

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160Polyamine Mathaba Gateway 2008 7 days Water F 1 #1Polyamine Mathaba UV_VIS_2 mAU

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7 8 9 10 1112

WVL:254 nm

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 25.0

-20 50 100 150 200

250Polyamine Mathaba Gateway 2008 7 days Water SC 1 #1Polyamine Mathaba UV_VIS_2 mAU

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5 6 7 8 9 10

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12 13 14

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16 17

18 19 20 21 2223 24

25 26

WVL:254 nm

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0

-100 250 500 750

1,200Polyamine Mathaba Gateway 2008 7 days Water MC 1 #1 UV_VIS_2 mAU

min 1 2

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5 6 7 8

9 10 11

12 13 14

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WVL:254 nm

(A) Standards

(D) Membrane conjugates (C) Soluble conjugates

(B) Free soluble

Time (min)

Fig 1:

151 0

40 80 120 0 40 80 120 160 200 240 0 1000 2000 3000 4000 5000 6000

Sun Valley 2007 Eston Estates 2008 Ukulinga 2007 Ukulinga 2008

Free soluble Soluble conjugates Membrane conjugates Putrescine (nmol g-1 DM)Spermidine (nmol g-1 DM)Spermine (nmol g-1 DM)

a

c b c

a

d

b c

b c b a

a a a a

b a b a

a a b a

b a b b

ab b aba

b b b a

A

C B

Fig 2:

152 Fig 3:

153 Fig 4:

154

0 1 2 3 4

Sun Valley Estates 2007

Eston Estates 2007 Ukulinga

2008 Ukulinga

2007

b

b a

a

Flavedo protein (mg g-1 DM)

Fig 5

155

Fig 6

156 Table 2.

Cold storage time (days)

Treatments Ukulinga 2007 Sun Valley Estates 2007

Free-soluble* Soluble-conjugates* Membrane-conjugates* Free soluble* Soluble conjugates* Membrane conjugates*

Put Spd Spm Put Spd Spm Put Spd Spm Put Spd Spm Put Spd Spm Put Spd Spm

Water dip (25^OC) 446 27.7 31.8 4144 13.9 87.5 35 54.8 108.3 2853 3.8 87.3 3711 25.7 105.3 5200 206.3 19.9

HW 47^oC 124 7.0 15.5 3135 12.4 33.4 25 34.8 252.2 2095 10.2 50.3 757 47.4 245.2 2793 199.8 281.0

0 HW 53^oC 254 26.2 130.6 4573 58.1 64.4 37 52.8 152.9 501 2.1 4.5 4279 84.5 130.4 1828 108.5 152.9

1 µM Mo 80 4.6 46.1 5596 33.6 300.7 44 13.7 101.4 3402 8.4 108.5 619 17.5 255.8 3337 341.9 152.1

1 µM Mo + HW 53^oC 990 115.0 390.8 7399 143.4 357.2 65 75.1 448.2 2304 22.0 72.4 1404 16.2 196.1 3104 61.3 93.7

10 µM Mo + HW 53^oC 60 2.9 3.3 7827 68.7 135.7 61 72.4 70.8 2549 41.5 74.1 3501 64.5 372.2 1462 89.7 236.2

Water dip (25^OC) 280 31.5 127.4 2083 7.1 262.9 18 39.8 127.5 2206 46.6 53.6 4495 41.2 201.1 4229 77.0 376.9

HW 47^oC 114 7.7 5.2 7071 122.0 1028 55 13.3 108.8 503 143.8 47.7 10216 169.0 600.7 9158 157.8 665.5

7 HW 53^oC 36 6.6 16.6 3400 92.6 488.6 26 39.9 62.5 2989 43.5 256.4 2242 242.6 388.8 803 67.6 62.5

1 µM Mo 745 60.7 211.5 6505 18.7 525.6 56 54.8 246.1 2425 42.7 71.4 2780 165.4 94.0 2600 29.5 502.5

1 µM Mo + HW 53^oC 812 52.0 163.9 1756 37.6 129.5 20 47.6 140.4 2600 28.9 222.3 323 15.2 101.1 1620 53.4 189.1

10 µM Mo + HW 53^oC 375 32.6 90.7 9769 592.0 644.9 78 196.7 469.1 3254 6.6 284.9 6908 273.0 133.2 2150 333.7 57.3

Water dip (25^OC) 879 45.7 147.9 3862 63.6 191.0 37 14.0 34.0 4686 56.0 134.4 629 367.8 264.0 1519 15.2 11.4

HW 47^oC 1131 194.7 371.8 1034 28.0 244.8 17 24.6 90.2 2595 32.0 187.6 6295 294.4 164.9 1700 8.10 83.2

14 HW 53^oC 365 19.2 45.1 1822 87.6 227.7 19 91.7 162.1 4174 27.0 33.6 3074 151.9 159.9 1447 46.5 162.1

1 µM Mo 470 33.0 25.3 701 91.2 88.6 9 17.4 281.7 4227 4.3 4.4 5869 29.0 328.0 4116 25.2 34.5

1 µM Mo + HW 53^oC 527 40.0 65.9 5392 31.9 83.4 46 32.4 71.3 151 4.8 7.0 405 29.4 272.2 3982 92.0 47.8

10 µM Mo + HW 53^oC 1073 27.7 62.2 331 15.1 121.5 11 34.2 175.3 4107 17.4 68.0 6785 35.9 444.5 331 27.0 132.6

Water dip (25^OC) 1516 15.8 80.6 11991 138.6 512.6 104 8.80 61.2 1917 66.0 94.9 15906 97.3 212.0 1060 96.4 5.8

HW 47^oC 529 79.6 146.7 13277 83.0 152.2 106 37.0 78.0 3643 28.4 10.1 1974 17.7 25.3 47 5.8 11.9

21 HW 53^oC 427 32.6 14.0 2466 27.9 45.1 22 50.1 477.0 1784 3.1 52.8 1343 24.7 79.1 42 4.6 477.0

1 µM Mo 272 22.3 21.6 9295 214.5 106.6 74 207.7 139.0 1016 17.4 149.9 3117 10.9 114.2 2442 55.9 119.0

1 µM Mo + HW 53^oC 489 61.0 84.5 4958 37.7 156.2 42 39.7 53.5 3401 20.3 19.4 767 82.2 73.8 2941 104.7 292.9

10 µM Mo + HW 53^oC 637 37.3 144.9 4710 86.6 113.1 41 63.5 122.9 2052 58.9 63.5 2188 49.6 108.3 186 156.8 41.4

Water dip (25^OC) 255 44.6 68.6 12749 19.7 187.2 100 69.5 373.1 2648 6.9 24.6 9791 190.8 150.8 2925 54.8 188.2

HW 47^oC 788 79.5 45.1 7152 20.6 124.9 61 101.3 370.5 4174 59.0 56.6 3963 370.3 646.1 2463 109.4 97.9

28 HW 53^oC 895 32.1 7.5 5643 167.9 230.2 50 26.4 298.6 4244 20.5 74.3 4545 133.8 150.2 2453 21.7 298.6

1 µM Mo 64 4.5 10.2 13753 54.5 73.9 106 54.3 252.0 5161 14.8 114.4 2937 154.1 120.9 44 60.6 459.9

1 µM Mo + HW 53^oC 279 24.2 10.7 7014 37.8 69.2 56 72.9 223.5 2878 8.9 233.2 806 61.4 218.6 885 95.4 100.5

10 µM Mo + HW 53^oC 583 33.0 34.7 9080 84.4 103.4 74 51.5 85.2 4408 90.1 105.7 3991 27.2 66.8 324 27.1 127.9

F prob (5%) P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001

*PAs - mmol g^-1 D

157 Table 3.

Cold storage time (days)

Treatments Ukulinga 2008 Eston Estates 2008

Free-soluble* Soluble-conjugates* Membrane-conjugates* Free-soluble* Soluble-conjugates* Membrane-conjugates*

Put Spd Spm Put Spd Spm Put Spd Spm Put Spd Spm Put Spd Spm Put Spd Spm

Water dip (25^OC) 388 46.3 59.3 4629 14.2 84.6 738 29.4 198.0 961 11.7 67.9 1934 53.9 28.9 358 16.2 65.1

HW 47^oC 411 20.2 147.7 3723 30.4 191.8 194 4.1 241.4 839 15.8 7.5 529 12.6 153.8 492 15.9 56.6

0 HW 53^oC 521 55.9 17.1 2390 316.6 396.4 604 125.1 606.8 850 17.6 39.3 1578 67.2 133.9 984 61.0 111.6

1 µM Mo 354 25.4 38.3 1820 265.4 59.1 509 27.5 166.4 924 50.0 102.9 2507 251.0 66.6 331 17.2 134.4

1 µM Mo + HW 53^oC 652 10.2 24.3 3519 44.6 40.3 680 67.5 152.9 2097 40.9 107.8 1510 4.0 84.4 466 83.5 110.9

10 µM Mo + HW 53^oC 71 8.2 9.0 3744 65.2 27.6 547 94.5 91.8 21.00 3.9 13.1 2846 80.7 11.4 1462 89.7 250.8

Water dip (25^OC) 590 31.0 151.8 1813 350.7 42.5 557 14.1 126.7 1038 36.6 121.3 4934 17.5 112.0 1621 49.4 110.2

HW 47^oC 115 4.2 32.5 417 108.9 244.5 262 7.7 0.80 49 3.5 13.2 1451 12.6 20.6 72 2.6 0.90

7 HW 53^oC 1275 12.4 177.5 2744 87.2 276.5 612 20.1 46.7 1317 9.6 82.0 3407 58.5 68.3 1914 100.2 16.5

1 µM Mo 692 55.6 114.8 2058 293.2 183.0 570 55.1 319.9 2084 65.5 65.7 296 142.5 12.0 467 50.0 266.8

1 µM Mo + HW 53^oC 81 1.2 2.6 6304 85.9 203.4 526 18.2 67.9 1331 9.6 26.0 3482 42.0 134.7 1871 33.9 226.0

10 µM Mo + HW 53^oC 150 2.7 17.5 3980 126.6 69.8 941 102.8 177.8 355 29.1 2.8 1340 139.3 24.0 2150 333.7 49.8

Water dip (25^OC) 484 2.3 16.3 1845 7.8 84.0 381 55.5 314.7 644 3.0 10.3 1667 117.6 21.7 531 4.6 14.6

HW 47^oC 403 37.8 31.2 2284 97.8 181.6 476 60.1 37.8 594 5.1 19.6 2621 79.0 30.1 663 55.5 188.9

14 HW 53^oC 512 20.5 33.6 1344 103.4 132.6 855 95.4 457.9 515 5.4 19.5 4074 72.8 12.8 684 8.1 205.3

1 µM Mo 30 13.9 1.3 1772 150.8 63.2 566 65.9 260.3 380 45.8 46.6 2482 309.7 127.0 1376 97.2 45.0

1 µM Mo + HW 53^oC 911 41.8 47.2 1358 79.6 38.5 425 14.6 91.6 530 6.8 39.7 1705 44.8 34.4 575 55.6 99.2

10 µM Mo + HW 53^oC 391 34.0 142.6 5064 49.6 158.2 727 34.5 192.6 1766 17.6 77.0 848 368.4 82.4 331 27.0 219.7

Water dip (25^OC) 200 33.1 28.9 1831 25.5 191.8 667 50.2 175.8 1816 15.8 29.6 674 147.1 27.6 477 197.4 317.3

HW 47^oC 1023 45.8 290.1 4024 31.2 394.8 346 10.5 85.8 1912 89.0 24.9 739 56.6 414.1 353 40.6 335.0

21 HW 53^oC 548 30.3 51.1 4599 78.5 463.5 597 60.4 9.2 767 34.8 15.9 654 29.6 651.3 292 41.7 151.0

1 µM Mo 626 60.5 125.3 2887 32.0 310.6 1552 36.9 65.8 2361 44.2 194.4 444 41.8 532.4 315 47.7 123.7

1 µM Mo + HW 53^oC 353 50.0 12.1 2594 5.0 88.9 280 60.5 123.8 787 27.3 49.9 3066 7.2 711.8 250 51.7 193.3

10 µM Mo + HW 53^oC 1268 112.5 37.0 3097 88.3 407.3 532 52.2 39.3 1800 13.1 15.3 578 132.9 230.1 186 156.8 324.4

Water dip (25^OC) 564 9.0 8.8 1011 61.0 58.1 400 12.7 19.3 784 20.0 45.2 1549 32.8 330.0 249 23.7 32.1

HW 47^oC 441 17.2 128.9 6855 231.8 385.4 1156 179.2 241.3 1196 24.0 445.2 1128 38.1 175.5 395 54.6 68.1

28 HW 53^oC 152 37.2 145.2 5469 133.9 120.3 387 79.0 272.2 443 3.1 49.1 508 785.5 253.7 215 49.9 41.0

1 µM Mo 890 57.6 121.8 4526 49.0 160.9 865 94.6 116.1 576 71.9 121.1 859 24.1 380.9 123 39.2 9.3

1 µM Mo + HW 53^oC 332 39.4 151.1 3268 48.1 105.5 418 47.7 204.4 912 8.3 149.0 748 28.4 311.7 190 61.3 194.4

10 µM Mo + HW 53^oC 1025 12.9 99.3 3385 22.7 285.9 709 56.3 127.4 528 28.6 225.1 1401 194.3 108.9 324 27.1 30.0

F prob (5%) P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001 P<.001

*PAs - mmol g^-1 DM

158